The present invention relates to polynucleotide reagents for the determination of target nucleotide sequences, and to diagnostic kits and methods employing such polynucleotide reagents.
A group of six inventors, including the present applicants, have described in applications U.S. Ser. No. 607,885, filed May 7, 1984, 684,305, filed Dec. 20, 1984, and 684,308, filed Dec. 20, 1984, reagent complexes and their use in diagnostic kits and methods. Such reagent complexes contain a probe polynucleotide (P) having a target binding region (TBR) substantially complementary to the target nucleotide sequence being determined, and a labeled polynucleotide (L) or signal strand (SS) as it is referred to in later applications, bound by complementary base pairing to at least some of the nucleotides of the target binding region (TBR) of the probe polynucleotide (P). In the method, a sample polynucleotide (G) displaces the signal strand from the reagent complex. Frequently, after a separation, the displaced signal strand (or labeled polynucleotide) is detected.
There is a brief discussion in each of U.S. Ser. No. 607,885, 684,305 and 684,308 of detecting two different target nucleotide sequences in a sample by employing two different reagent complexes, each with a unique label, and each with a target binding region substantially complementary to one of the two target nucleotide sequences.
Sandwich polynucleotide assays have been described, e.g., in U.S. Pat. No. 4,486,539 (1984), in which two probe polynucleotides are used, each complementary to a portion of the target nucleotide sequence. Sample polynucleotide having such a target nucleotide sequence binds to both probes, one immobilized, the other in solution and bearing a label, e.g., a radiolabel. After washing, the detectable label present on the surface is intended to be limited to those labeled probes bound via sample to the immobilized probes. Again, the two portions of the target nucleotide sequence are complementary to sequences of two separate probe polynucleotides. See also EPA No. 139,489 (May 2, 1985).